Comparison of non-magnetic and magnetic beads multiplex assay for assessment of Plasmodium falciparum antibodies

Background New reagents have emerged allowing researchers to assess a growing number of vaccine-associated immune parameters. Multiplex immunoassay(s) are emerging as efficient high-throughput assays in malaria serology. Currently, commercial vendors market several bead reagents for cytometric bead assays (CBA) but relative performances are not well published. We have compared two types of bead-based multiplex assays to measure relative antibody levels to malarial antigens. Methods Assays for the measurement of antibodies to five Plasmodium falciparum vaccine candidates using non-magnetic and magnetic fluorescent microspheres were compared for their performances with a Bio-Plex200 instrument. Mean fluorescence intensity (MFI) was determined from individuals from western Kenya and compared to known positive and negative control plasma samples. Results P. falciparum recombinant antigens were successfully coupled to both non-magnetic and magnetic beads in multiplex assays. MFIs between the two bead types were comparable for all antigens tested. Bead recovery was superior with magnetic beads for all antigens. MFI values of stored non-magnetic coupled beads did not differ from freshly coupled beads, though they showed higher levels of bead aggregation. Discussion Magnetic and non-magnetic beads performed similarly in P. falciparum antibody assays. Magnetic beads were more expensive, but had higher bead recovery, were more convenient to use, and provided rapid and easy protocol manipulation. Magnetic beads are a suitable alternative to non-magnetic beads in malarial antibody serology

Stabilization of RDT target antigens present in dried Plasmodium falciparum-infected samples for validating malaria rapid diagnostic tests at the point of care

Abstract Background Malaria rapid diagnostic tests (RDTs) are a great achievement in implementation of parasite based diagnosis as recommended by World Health Organization. A major drawback of RDTs is lack of positive controls to validate different batches/lots at the point of care. Dried Plasmodium falciparum-infected samples with the RDT target antigens have been suggested as possible positive control but their utility in resource limited settings is hampered by rapid loss of activity over time. Methods This study evaluated the effectiveness of chemical additives to improve long term storage stability of RDT target antigens (HRP2, pLDH and aldolase) in dried P. falciparum-infected samples using parasitized whole blood and culture samples. Samples were treated with ten selected chemical additives mainly sucrose, trehalose, LDH stabilizer and their combinations. After baseline activity was established, the samples were air dried in bio-safety cabinet and stored at room temperatures (~ 25 °C). Testing of the stabilized samples using SD Bioline, BinaxNOW, CareStart, and First Response was done at intervals for 53 weeks. Results Stability of HRP2 at ambient temperature was reported at 21–24 weeks while that of PAN antigens (pLDH and aldolase) was 2–18 weeks of storage at all parasite densities. The ten chemical additives increased the percentage stability of HRP2 and PAN antigens. Sucrose alone and its combinations with Alsever’s solution or biostab significantly increased stability of HRP2 by 56% at 2000 p/µL (p < 0.001). Trehalose and its combinations with biostab, sucrose or glycerol significantly increased stability of HRP2 by 57% (p < 0.001). Unlike sucrose, the stability of the HRP2 was significantly retained by trehalose at lower concentrations (500, and 200 p/µL). Trehalose in combination biostab stabilizer increased the percentage stability of PAN antigens by 42, and 32% at 2000 and 500 p/µL respectively (p < 0.01). This was also the chemical combination with the shortest reconstitution time (~ < 20 min). Conclusions These findings confirm that stabilizing RDT target antigens in dried P. falciparum-infected samples using chemical additives provides field-stable positive controls for malaria RDTs

Interferon-γ responses to Plasmodium falciparum vaccine candidate antigens decrease in the absence of malaria transmission

Background Malaria elimination campaigns are planned or active in many countries. The effects of malaria elimination on immune responses such as antigen-specific IFN- γ responses are not well characterized. Methods IFN- γ responses to the P. falciparum antigens circumsporozoite protein, liver stage antigen-1, thrombospondin-related adhesive protein, apical membrane antigen-1, MB2, and merozoite surface protein-1 were tested by ELISA in 243 individuals in highland Kenya in April 2008, October 2008, and April 2009, after a one-year period of interrupted malaria transmission from April 2007 to March 2008. Results While one individual (0.4%) tested positive for P. falciparum by PCR inOctober 2008 and another two (0.9%) tested positive in April 2009, no clinical malaria cases were detected during weekly visits. Levels of IFN-γ to all antigens decreased significantly from April 2008 to April 2009 (all P < 0.001). Discussion Naturally acquired IFN- γ responses to P. falciparum antigensare short-lived in the absence of repeated P. falciparum infection. Even short periods of malaria interruption may significantly decrease IFN-γ responses to P. falciparum antigens

Distributed under Creative Commons CC-BY 4.0 Interferon-γ responses to Plasmodium falciparum vaccine candidate antigens decrease in the absence of malaria transmission

ABSTRACT Background. Malaria elimination campaigns are planned or active in many countries. The effects of malaria elimination on immune responses such as antigen-specific IFN-γ responses are not well characterized. Methods. IFN-γ responses to the P. falciparum antigens circumsporozoite protein, liver stage antigen-1, thrombospondin-related adhesive protein, apical membrane antigen-1, MB2, and merozoite surface protein-1 were tested by ELISA in 243 individuals in highland Keny

Changes in B Cell Populations and Merozoite Surface Protein-1-Specific Memory B Cell Responses after Prolonged Absence of Detectable <i>P. falciparum</i> Infection

<div><p>Clinical immunity to malaria declines in the absence of repeated parasite exposure. However, little is known about how B cell populations and antigen-specific memory B cells change in the absence of <i>P. falciparum</i> infection. A successful indoor residual insecticide spraying campaign in a highland area of western Kenya, led to an absence of blood-stage <i>P. falciparum</i> infection between March 2007 and April 2008. We assessed memory B cell responses in 45 adults at the beginning (April 2008) and end (April 2009) of a subsequent 12-month period during which none of the adults had evidence of asymptomatic parasitemia or clinical disease. Antibodies and memory B cells to the 42-kDa portion of the merozoite surface protein-1 (MSP-1₄₂) were measured using ELISA and ELISPOT assays, respectively. B cell populations were characterized by flow cytometry. From 2008 to 2009, the prevalence of MSP-1₄₂-specific memory B cells (45% vs. 55%, respectively, <i>P</i> = 0.32) or antibodies (91% vs. 82%, respectively, <i>P</i> = 0.32) did not differ significantly, although specific individuals did change from positive to negative and vice versa, particularly for memory B cells, suggesting possible low-level undetected parasitemia may have occurred in some individuals. The magnitude of MSP-1₄₂-specific memory B cells and levels of antibodies to MSP-1₄₂ also did not differ from 2008 to 2009 (<i>P</i>>0.10 for both). However, from 2008 to 2009 the proportions of both class-switched atypical (CD19+IgD-CD27-CD21-IgM-) and class-switched activated (CD19+IgD-CD27+CD21-IgM-) memory B cells decreased (both <i>P</i><0.001). In contrast, class-switched resting classical memory B cells (CD19+IgD-CD27+CD21+IgM-) increased (<i>P</i><0.001). In this area of seasonal malaria transmission, a one- year absence of detectable <i>P. falciparum</i> infection was not associated with changes in the prevalence or level of MSP-1₄₂ specific memory B cells, but was associated with major changes in overall memory B cell subsets.</p></div

MOESM1 of Stabilization of RDT target antigens present in dried Plasmodium falciparum-infected samples for validating malaria rapid diagnostic tests at the point of care

Additional file 1. Percentage stability of Plasmodium HRP2, pLDH, and aldolase after 53 weeks of storage tested on three malaria RDTs after stabilization using chemical additives

The proportions of marginal zone (CD19+IgD+CD27+IgM+) B cells increased between 2008 and 2009.

<p>The proportions of marginal zone (CD19+IgD+CD27+IgM+) B cells increased between 2008 and 2009.</p

Prevalence of toxoid (TT)-specific immunoglobulin G (IgG) antibodies in highland Kenyan adults in 2008 and 2009.

<p>McNemar’s test, <i>P</i> = 1.0.</p

Comparison between estimated memory B cell frequencies and antibody responses to merozoite surface protein-1₄₂ (MSP-1₄₂) and tetanus toxoid (TT).

<p>There was no association between specific MBCs and antibodies for both MSP-1₄₂ and TT in 2008 and 2009.</p

Number and percentages of responders to tetanus toxoid (TT) as assessed by specific memory B cells and immunoglobulin G (IgG) antibodies (Ab) in April 2009.

<p>McNemar’s test, <i>P = </i>0.71.</p